1. Field of the Invention
This invention relates to an efficient method for refolding insulin-like growth factor-I (IGF-I) that has been produced in heterologous host cells and is present in these cells as clumps of insoluble protein.
2. Description of Related Art
The production of large quantities of relatively pure, biologically active polypeptides and proteins is important economically for the manufacture of human and animal pharmaceutical formulations, enzymes, and other specialty chemicals. For production of many proteins, recombinant DNA techniques have become the method of choice because large quantities of exogenous proteins can be expressed in bacteria and other host cells free of other contaminating proteins.
Producing recombinant protein involves transfecting host cells with DNA encoding the protein and growing the cells under conditions favoring expression of the recombinant protein. The prokaryote E. coli is favored as host because it can be made to produce recombinant proteins in high yields. Numerous U.S. patents on general bacterial expression of recombinant-DNA-encoded proteins exist, including U.S. Pat. Nos. 4,565,785 on a recombinant DNA molecule comprising a bacterial gene for an extracellular or periplasmic carrier protein and non-bacterial gene; 4,673,641 on coproduction of a foreign polypeptide with an aggregate-forming polypeptide; 4,738,921 on an expression vector with a trp promoter/operator and trp LE fusion with a polypeptide such as IGF-I; 4,795,706 on expression control sequences to include with a foreign protein; and 4,710,473 on specific circular DNA plasmids such as those encoding IGF-I.
Under some conditions, certain heterologous proteins expressed in large quantities from bacterial hosts are precipitated within the cells in dense masses, recognized as bright spots visible within the enclosure of the cells under a phase contrast microscope at magnifications down to 1000 fold. These clumps of precipitated proteins are referred to as "refractile bodies," and constitute a significant portion of the total cell protein. Brems et al., Biochemistry, 24: 7662 (1985). On the other hand, the clumps of protein may not be visible under the phase contrast microscope, and the expression "inclusion body" is often used to refer to the aggregates of protein whether visible or not under the phase contrast microscope.
Recovery of the protein from these bodies has presented numerous problems, such as how to separate the protein encased within the cell from the cellular material and proteins harboring it, and how to recover the inclusion body protein in biologically active form. The recovered proteins are often biologically inactive because they are folded into a three-dimensional conformation different from that of active protein. For example, recombinant IGF-I that has disulfide bonds formed between cysteine pairs different from the pairs found in the disulfide bonds of native IGF-I has significantly reduced biological activity. Raschdorf et al., Biomedical and Environmental Mass Spectroscopy, 16: 3-8 (1988). Misfolding occurs either in the cell or during the isolation procedure. Methods for refolding the proteins into the correct, biologically active conformation are essential for processing functional proteins.
Protein folding is influenced by the nature of the medium containing the protein and by a combination of weak attractive or repellent intramolecular forces involved in hydrogen bonding, ionic bonding, and hydrophobic interactions. When pairs of cysteine residues are brought into close proximity as the peptide backbone folds, strong covalent disulfide bonds form between cysteine residues, serving to lock the tertiary conformation in place. Refolding protocols have been designed to break incorrect disulfide bonds, block random disulfide bonding, and allow refolding and correct disulfide bonding under conditions favorable to the formation of active conformer.
It has been found that the soluble proportion of high-level expressed protein in E. coli has been dramatically increased by lowering the temperature of fermentation to below 30.degree. C. A considerable fraction of various foreign proteins, i.e., human IFN-.alpha.2, IFN-.gamma., and murine MX protein [Schein and Noteborn, Bio/Technology, 6: 291-294 (1988)] and human IFN-.beta. [Mizukami et al., Biotechnol. Lett., 8: 605-610 (1986)], stayed in solution. This procedure represents an alternative to renaturation of proteins recovered from refractile bodies, but requires an expression system that is efficiently induced at temperatures below 30.degree. C. The procedure is therefore not effective for all proteins.
One series of techniques for recovering active protein from inclusion bodies involves solubilizing the inclusion bodies in strongly denaturing solutions and then optionally exchanging weakly denaturing solutions for the strongly denaturing solutions (or diluting the strong denaturant), or using molecular sieve or high-speed centrifugation techniques. Such recovery methods, described, e.g., in U.S. Pat. Nos. 4,512,922; 4,518,256; 4,511,502; and 4,511,503, are regarded as being universally applicable, with only minor modifications, to the recovery of biologically active recombinant proteins from inclusion bodies. These methods seek to eliminate random disulfide bonding prior to coaxing the recombinant protein into its biologically active conformation through its other stabilizing forces.
In one such method, the denatured protein desired to be refolded is further purified under reducing conditions that maintain the cysteine moieties of the protein as free sulfhydryl groups by supplying a reducing agent throughout all the purification steps. This allows the protein to refold itself under the conditions of purification in the absence of incorrect disulfide bond formation. The reducing agent is then diluted into an aqueous solution to enable the refolded protein to form the appropriate disulfide bonds in the presence of air or some other oxidizing agent. This enables refolding to be easily incorporated into the overall purification process and works optimally for recombinant proteins having relatively uncomplicated tertiary structures in their biologically active forms.
In a second approach in this series, refolding of the recombinant protein takes place in the presence of both the reduced (R--SH) and oxidized (R--S--S--R) forms of a sulfhydryl compound. This allows free sulfhydryl groups and disulfides to be formed and reformed constantly throughout the purification process. The reduced and oxidized forms of the sulfhydryl compound are provided in a buffer having sufficient denaturing power that all of the intermediate conformations of the protein remain soluble in the course of the unfolding and refolding. Urea is suggested as a suitable buffer medium because of its apparent ability to act both as a sufficiently weak denaturing agent to allow the protein to approximate its correct conformation and as a sufficiently strong denaturant that the refolding intermediates maintain their solubility. This approach works best where the recombinant inclusion body proteins of interest have relatively uncomplicated folding patterns.
The third alternative in this series, used in more difficult refolding situations, is designed to break any disulfide bonds that may have formed incorrectly during isolation of the inclusion bodies and then to derivatize the available free sulfhydryl groups of the recombinant protein. This objective is achieved by sulfonating the protein to block random disulfide pairings, allowing the protein to refold correctly in weak denaturant, and then desulfonating the protein, which protocol favors correct disulfide bonding. The desulfonation takes place in the presence of a sulfhydryl compound and a small amount of its corresponding oxidized form to ensure that suitable disulfide bonds will remain intact. The pH is raised to a value such that the sulfhydryl compound is at least partially in ionized form to enhance nucleophilic displacement of the sulfonate.
These refolding protocols, while practical for their universal utility, have not been shown necessarily to be maximally efficient with, for example, recombinant IGF-I.
Enhancement of selected disulfide pairings by adding 50% methanol to buffer at low ionic strength has been reported by G. H. Snyder, J. Biol. Chem., 259: 7468-7472 (1984). The strategy involves enhancing formation of specific disulfide bonds by adjusting electrostatic factors in the medium to favor the juxtaposition of oppositely charged amino acids that border the selected cysteine residues. See also Tamura et al., abstract and poster presented at the Eleventh American Peptide Symposium on Jul. 11, 1989 advocating addition of acetonitrile, DMSO, methanol, or ethanol to improve the production of the correctly folded IGF-I.
U.S. Pat. No. 4,923,967 and EP 361,830 describe a protocol for solubilizing and sulphitolysing refractile protein in denaturant, then exchanging solvent to precipitate the protein. The protein is resolubilized in denaturant and allowed to refold in the presence of reducing agent. The multiple steps required to achieve correct folding are time-consuming.
The recovery of the biological activity requires a carefully monitored renaturation procedure and may be very difficult depending on the protein in question. A number of publications have appeared that report refolding attempts for individual proteins that are produced in bacterial hosts or are otherwise in a denatured or non-native form. For example, formation of a dimeric, biologically active M-CSF after expression in E. coli is described in WO 88/8003 and by Halenbeck et al., Biotechnology, 7: 710-715 (1989). The procedures described involve the steps of initial solubilization of M-CSF monomers isolated from inclusion bodies under reducing conditions in a chaotropic environment comprising urea or guanidine hydrochloride, refolding achieved by stepwise dilution of the chaotropic agents, and final oxidation of the refolded molecules in the presence of air or a redox-system.
Reasonable recovery after renaturation has been reported for several proteins such as interleukin-2 (IL-2) [Tsuji et al., Biochemistry, 26: 3129-3134 (19871) WO 88/8849], growth hormone from various sources [George et al., DNA, 4: 273-281 (1984); Gill et al., Bio/Technology, 3: 643-646 (1985); Sekine et al., Proc. Natl. Acad. Sci. USA, 82: 4306-4310 (1985); U.S. Pat. No. 4,985,544, the lattermost reference involving adding a denaturing agent and reducing agent to solubilize the protein, removing the reducing agent, oxidizing the protein, and removing the denaturing agent], prochymosin [Green et al., J. Dairy Res., 52: 281-286 (1985)], urokinase [Winkler et al., Bio/Technology, 3: 990-1000 (1985)], somatotropin [U.S. Pat. No. 4,652,630, whereby urea is used for solubilization, and a mild oxidizing agent is then used for refolding], and tissue-plasminogen activator [Rudolph et al., in "623rd Biochem. Soc. Meeting," Canterbury (1987)] . See also Marston, Biochemical J., 240: 1 (1986).
Where the efficiency of recovery has been reported, up to 40% active foreign protein has been obtained. See, e.g, Boss et al., Nucl. Acids Res., 12: 3791-3806 (1984); Cabilly et al., Proc. Natl. Acad. Sci. USA, 81: 3273-3277 (1984); Marston et al., Bio/Technology, 2: 800-804 (1984); and Rudolph et al., supra. However, such yields may not be acceptable if the protein is costly to produce and must be made in commercial quantities.
Additional representative literature on refolding of nonnative proteins derived from different sources includes a report that IL-2 and interferon-.beta. (IFN-.beta.) have been refolded using SDS for solubilization and Cu.sup.+2 ions as oxidation promoters of the fully reduced proteins [U.S. Pat. No. 4,572,798]. A process for isolating recombinant refractile proteins as described in U.S. Pat. No. 4,620,948 involves using strong denaturing agents to solubilize the proteins, reducing conditions to facilitate correct folding, and denaturant replacement in the presence of air or other oxidizing agents to reform the disulfide bonds. The proteins to which the process can be applied include urokinase, human, bovine, and porcine growth hormone, interferon, tissue-type plasminogen activator, FMD coat protein, prorennin, and the src protein.
A method for renaturing unfolded proteins including cytochrome c, ovalbumin, and trypsin inhibitor by reversibly binding the denatured protein to a solid matrix and stepwise renaturing it by diluting the denaturant is disclosed in WO 86/5809. A modified monomeric form of human platelet-derived growth factor (PDGF) expressed in E. coli has been S-sulfonated during purification to protect thiol moieties and then dimerized in the presence of oxidizing agents to yield the active protein. Hoppe et al., Biochemistry, 28: 2956 (1989).
Additionally, EP 433,225 published 19 Jun. 1991 discloses a process for producing dimeric biologically active transforming growth factor-.beta. protein or a salt thereof wherein the denatured monomeric form of the protein is subjected to refolding conditions that include a solubilizing agent such as mild detergent, an organic, water-miscible solvent, and/or a phospholipid. See also Bowden et al., Bio/Technology, 9: 725 (1991) on .beta.-lactamase cytoplasmic and periplasmic inclusion bodies, and Samuelsson et al., Bio/Technology, 9: 731 (1991) on refolding of human interferon-gamma mutants. For general review articles, see Marston, Biochem. J., 240: 1-12 (1986); Mitraki and King, Bio/Technology, 7: 690 (1989); Marston and Hartley, Methods in Enzymol., 182: 264-276 (1990); Wetzel, "Protein Aggregation In Vivo: Bacterial Inclusion Bodies and Mammalian Amyloid," in Stability of Protein Pharmaceuticals: In Vivo Pathways of Degradation and Strategies for Protein Stabilization, Ahern and Manning (eds.), Plenum Press, 1991; and Wetzel, "Enhanced Folding and Stabilization of Proteins by Suppression of Aggregation In Vitro and In Vivo," in Protein Engineering--A Practical Approach, Rees, A. R. et al. (eds.), IRL Press at Oxford University Press, Oxford, 1991.
Several literature references exist on the production of IGF-I in bacteria. These include EP 128,733 published 19 Dec. 1984 and EP 135,094 published 27 Mar. 1985, which address expression of IGF-I in bacteria. EP 288,451 addresses use of lamB or ompF signal to secrete IGF-I in bacteria; Obukowicz et al., Mol. Gen. Genet., 215: 19-25 (1988) and Wong et al., Gene, 68: 193-203 (1988) teach similarly. EP 286,345 discloses fermentation of IGF-I using a lambda promoter.
In addition, methods have been suggested for preparing IGF-I as a fusion protein. For example, EP 130,166 discloses expression of fusion peptides in bacteria, .and EP 155,655 (U.S. Pat. No. 5,019,500) and EP 219,814 disclose a fusion of IGF-I with a protective polypeptide for expression in bacteria. EP 264,074 discloses a two-cistronic met-IGF-I expression vector with a protective peptide of 500-50,000 molecular weight [see also U.S. Pat. No. 5,028,531 and Saito et al., J. Biochem., 101: 1281-1288 (1987)]. Other IGF-I fusion techniques that have been reported include fusion with protective peptide from which a rop gene is cut off [EP 219,814], IGF-I multimer expression [Schulz et al., J. Bacteriol., 169: 5385-5392 (1987)], fusion of IGF-I with LH protein through a chemically cleavable methionyl or tryptophan residue at the linking site [Saito et al., J. Biochem., 101: 123-134 (1987)], and fusion with superoxide dismutase [EP 196,056]. Niwa et al., Ann. NY Acad. Sci., 469: 31-52 (1986) discusses the chemical synthesis, cloning, and successful expression of genes for IGF-I fused to another polypeptide.
These methods utilizing fusion proteins, however, generally require a relatively long leader sequence and are directed to improving expression of the inclusion body protein, not to improving refolding of the denatured recombinant protein. Int. Pub. No. WO 91/02807 published 7 Mar. 1991 describes a method for refolding recombinant IGF-I that involves cloning the IGF-I gene with a positively charged leader sequence prior to transfecting the DNA into the host cell. The additional positive charge on the amino terminus of the recombinant IGF-I allows correct refolding when the solubilized protein is stirred for 2-16 hours in denaturant solution. Following refolding, the leader sequence is cleaved and the active recombinant protein is purified. This multistep process is burdensome, requiring additional materials and effort to clone a heterologous leader sequence in front of the IGF-I gene and then to remove the leader sequence from the purified protein. Additionally, the 30-50% yield of active conformer, using this method, is unremarkable.
Another method for facilitating in vitro refolding of recombinant IGF-I involves using a solubilized affinity fusion partner consisting of two IgG-binding domains (ZZ) derived from staphylococcal protein A. Samuelsson et al., Bio/Technology, 9: 363 (1991). While this method, which uses the protein A domain as a solubilizer of misfolded and multimeric IGF-I, results in higher yields of IGF-I without the use of denaturing agents or redox chemicals, it involves the extra steps of fusing onto the IGF-I gene a separate gene and removing the polypeptide encoded by that gene after expression of the fusion gene.
As regards the bacterial hosts that may be utilized for fermentation processes, WO 88/05821 published 11 Aug. 1988 discloses a method of isolating a mutant strain of E. coli having a defective periplasmic protease. WO 89/02465 published 23 Mar. 1989 discloses a process for production of a polypeptide comprising direct expression of the polypeptide in bacterial host cells using an inducible expression system in combination with a protease-deficient bacterial host system, including a host deficient in two proteases. WO 85/03949 published 12 Sep. 1985 discloses bacterial cell strains carrying specific mutations within their DNA sequences that cause the cells to exhibit a reduced capacity for degrading foreign products due to reduced expression of cellular proteases, with a htpR lon E. coli host exemplified. WO 89/10976 published 16 Nov. 1989 discloses protease-deficient gram-positive bacteria and their use as host organisms for producing recombinant proteins. In addition, Buell et al., Nucl. Acids Res., 13: 1923-1938 (1985) discloses use of an E. coli host mutated at lon and htpR to produce IGF-I.
There is a need in the art for a simple, one-step, efficient protocol for refolding insoluble, misfolded IGF-I into the correct conformation so that the biological activity of the IGF-I can be restored.
Accordingly, it is an object of the present invention to provide such procedure for reactivating, in one step, misfolded IGF-I recovered from inclusion bodies formed in prokaryotic cells, allowing for recovery of biologically active IGF-I at low cost and high yield.
It is a particularly preferred object to provide a one-step solubilization and refolding procedure to reactivate misfolded recombinant IGF-I precipitated in the periplasmic space of bacterial host cells.
It is another object to provide protease-deficient E. coli hosts that are particularly suited for the solubilization and refolding process herein.
These and other objects will be apparent to those of ordinary skill in the art.